Pyruvate carboxylase from a thermophilic Bacillus. Studies on the specificity of activation by acyl derivatives of coenzyme A and on the properties of catalysis in the absence of activator

1. Oxaloacetate synthesis catalysed by pyruvate carboxylase from a thermophilic Bacillus in the absence of acetyl-CoA required addition of high concentrations of pyruvate, MgATP(2-) and HCO(3) (-), and at 45 degrees C occurred at a maximum rate approx. 20% of that in the presence of a saturating concentration of acetyl-CoA. The apparent K(m) for HCO(3) (-) at pH7.8 was 400mm without acetyl-CoA, and 16mm with a saturating activator concentration. The relationship between reciprocal initial rate and reciprocal MgATP(2-) concentration was non-linear (convex-down) in the absence of acetyl-CoA, but the extent of deviation decreased as the activator concentration was increased. The relationship between reciprocal initial rate and reciprocal pyruvate concentration was non-linear (convex-down) in the presence or absence of acetyl-CoA. 2. The optimum pH for catalysis of oxaloacetate synthesis was similar in the presence or absence of acetyl-CoA. The variation with pH of apparent K(m) for HCO(3) (-) implicated residue(s) with pK(a) 8.6 in catalysis of the activator-independent oxaloacetate synthesis. 3. Linear Arrhenius and van't Hoff plots were observed for the temperature-dependence of oxaloacetate synthesis in the absence of acetyl-CoA over the range 25-55 degrees C. E(a) (activation energy) was 56.3kJ/mol and DeltaH(double dagger) (HCO(3) (-)) (enthalpy of activation) was -38.6kJ/mol. In the presence of acetyl-CoA, biphasic Arrhenius and van't Hoff plots are observed with a change of slope at 30 degrees C in each case. E(a) was 43.7 and 106.3kJ/mol above and below 30 degrees C respectively. 4. Incubation of Bacillus pyruvate carboxylase with trinitrobenzenesulphonate caused specific inactivation of acetyl-CoA-dependent catalytic activity associated with the incorporation of 1.3+/-0.2 trinitrophenyl residues per subunit. Activator-independent catalysis and regulatory inhibition by l-aspartate were unaffected. The rate of inactivation of acetyl-CoA-dependent catalysis by trinitrobenzenesulphonate was specifically decreased by addition of acetyl-CoA and other acetyl-CoA and other acyl-CoA species, but complete protection was not obtained. 5. All alkylacyl derivatives of CoA tested activated Bacillus pyruvate carboxylase; acetyl-CoA was the most effective. The apparent K(a) exhibited a biphasic relationship with acyl-chain length for the straight-chain homologues. Certain long-chain acyl-CoA species showed additional activation at a high concentration. Weak activation occurred on addition of CoA or adenosine 3',5'-bisphosphate, but carboxyacyl-CoA species and derivatives containing a modified phosphoadenosyl group were inhibitory. Thioesters of CoA with non-carboxylic acids, e.g. methanesulphonyl-CoA, serve as activators of the thermophilic Bacillus and Saccharomyces cerevisiae pyruvate carboxylases, but as inhibitors of pyruvate carboxylases obtained from chicken and rat liver. 6. alpha-Oxoglutarate mimics the effect of l-aspartate as a regulatory inhibitor of the pyruvate carboxylases from both the thermophilic Bacillus and Saccharomyces cerevisiae. l-Glutamate was ineffective in both cases.     

Localization of vimentin,the nonspecific intermediate filament protein,in embryonal glia and in early differentiating neurons: In vivo and in vitro immunofluorescence study of the rat embryo with vimentin and neurofilament antisera

Antisera raised against vimentin, the protein subunit of nonspecific intermediate-sized filaments (IFs), were used in conjunction with neurofilament (NF) antisera to study the early development of neurons and glia in the rat embryo. Vimentin-positive fibers spanning the entire thickness of the neural tube including the cerebral vesicles were first observed on Day 12, concomitant with the appearance of NF protein in more confined areas (anterolateral regions of spinal cord and brain stem; motor roots emerging from the NF-positive areas). From Day 15 onwards vimentin and NF antisera selectively decorated glia and neurons, respectively, both in vivo and in vitro. Before Day 15 it appeared that NF-positive structures also stained with antivimentin in cryostat sections. In vitro experiments confirmed the presence of vimentin in early differentiating neurons. NF-positive cells were observed which also reacted with antivimentin in cultures obtained from 13- and 14-day embryos, but not later in development. Most neurons in these cultures became vimentin negative after 2–3 days in vitro.     

Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.     

Practitioners of vipassana meditation exhibit enhanced slow wave sleep and REM sleep states across different age groups

Sleep and Biological Rhythms - Intense meditation practices influence brain functions in different ways and at different levels. Earlier studies have shown that meditation practices help to...     

Chitotriosidase - a putative biomarker for sporadic amyotrophic lateral sclerosis

Background

Potential biomarkers to aid diagnosis and therapy need to be identified for Amyotrophic Lateral Sclerosis, a progressive motor neuronal degenerative disorder. The present study was designed to identify the factor(s) which are differentially expressed in the cerebrospinal fluid (CSF) of patients with sporadic amyotrophic lateral sclerosis (SALS; ALS-CSF), and could be associated with the pathogenesis of this disease.

Results

Quantitative mass spectrometry of ALS-CSF and control-CSF (from orthopaedic surgical patients undergoing spinal anaesthesia) samples showed upregulation of 31 proteins in the ALS-CSF, amongst which a ten-fold increase in the levels of chitotriosidase-1 (CHIT-1) was seen compared to the controls. A seventeen-fold increase in the CHIT-1 levels was detected by ELISA, while a ten-fold elevated enzyme activity was also observed. Both these results confirmed the finding of LC-MS/MS. CHIT-1 was found to be expressed by the Iba-1 immunopositive microglia.

Conclusion

Elevated CHIT-1 levels in the ALS-CSF suggest a definitive role for the enzyme in the disease pathogenesis. Its synthesis and release from microglia into the CSF may be an aligned event of neurodegeneration. Thus, high levels of CHIT-1 signify enhanced microglial activity which may exacerbate the process of neurodegeneration. In view of the multifold increase observed in ALS-CSF, it can serve as a potential CSF biomarker for the diagnosis of SALS.     

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.     

Evaluation of sleep architecture in practitioners of Sudarshan Kriya yoga and Vipassana meditation

Yoga is an ancient Indian science and way of life that has been described in the traditional texts as a systematic method of achieving the highest possible functional harmony between body and mind. Yogic practices are claimed to enhance the quality of sleep. Electrophysiological correlates associated with the higher states of consciousness have been reported in long-term practitioners of transcendental meditation during deep sleep states. The present study was carried out to assess sleep architecture in Sudarshan Kriya Yoga (SKY) and Vipassana meditators. This was to ascertain the differences, if any, in sleep architecture following yogic practices. Whole night polysomnographic recordings were carried out in 78 healthy male subjects belonging to control and yoga groups. The groups studied were aged between 20 and 30-years-old (younger) and 31 to 55-years-old (middle-aged). The sleep architecture was comparable among the younger control and yoga groups. While slow wave sleep (non-REM (rapid eye movement) S₃ and S₄) had reduced to 3.7 percent in the middle-aged control group, participants of the middle-aged yoga groups (both SKY and Vipassana) showed no such decline in slow wave sleep states, which was experienced by 11.76 and 12.76 percent, respectively, of the SKY and Vipassana groups. However, Vipassana practitioners showed a significant enhancement (P < 0.001) in their REM sleep state from that of the age-matched control subjects and also from their SKY counterparts. Yoga practices help to retain slow wave sleep and enhance the REM sleep state in the middle age; they appear to retain a younger biological age as far as sleep is concerned. Overall, the study demonstrates the possible beneficial role of yoga in sleep-wakefulness behavior.